S-Adenosylhomocysteine hydrolase is the only enzyme in mammalian cells for the removal of adenosylhomocysteine, the end-product of biological methylation reactions. For this reason, the enzyme is critical for the regulation of adenosylmethionine-dependent methylations. We have used several approaches to investigate structure/function relationships of AdoHcy hydrolase. We have cloned AdoHcy hydrolase cDNA from 5 species, and shown that AdoHcy hydrolases are expressed efficiently in E. coli. The amino acid sequences are highly conserved between species (64% identity between rat and Rhodobacter sequences) suggesting that much of the hydrolase sequence is required for enzyme function. The Rhodobacter sequence has an additional 36 amino acid segment that is not found in hydrolases from the animal kingdom. Expression of the rat liver enzyme in a Rhodobacter mutant lacking a functional hydrolase restores photopigment synthesis and photosynthetic growth. This experiment indicates that the 36 amino acid segment does not have a specific function in photosynthetic growth, and adds weight to the argument that the additional segment is a happenstance of evolution. Site-directed mutagenesis and protein modification studies of rat AdoHcy hydrolase have allowed us to assign amino acids 213 to 244 a role in the binding of NAD, and Cys78, Cys112 and Cys52 a location near the AdoHcy binding site. These studies have also indicated that amino acids 1 to 112 are less tightly folded than the remainder of the hydrolase polypeptide. Mammalian AdoHcy hydrolase is expressed at a significantly higher level in liver compared to most other tissues, indicating transcriptional control of expression. The hydrolase gene has been cloned, and the 10 exons, 9 introns, 1.2 kb of the 5'-flanking region and 0.5 kb of the 3'- flanking region have all been completely sequenced. The 5' end of the gene has the properties of a housekeeping gene in that transcriptions tarts from multiple sites, the sequence is highly enriched in CpG, and a TATA-like sequence, TATTTAAA, is found 23 bp upstream from the first transcriptional start site. Deletion analysis utilizing a reporter plasmid containing the 5'-flanking region has identified an Sp1 sequence critical for expression.